This project seeks to define the control principles that determine the complex relationship between signal input and output function in mammalian cells, and ultimately to generate quantitative computational models to describe cellular behavior in circumstances relevant to infectious disease. Using macrophages as a model system, we are characterizing the cellular response both to pattern recognition receptor (PRR) ligands and also to intact pathogens by measurement of the cellular response through a variety of readouts such as;signaling protein phosphorylation, intracellular trafficking, pathogen replication, transcription and production of immune mediators. We have profiled the response of RAW264.7 murine macrophage cells to a group of 4 toll-like receptor (TLR) ligands (LPS, Pam2CSK4, Pam3CSK4 and Resiquimod 848). Despite these receptors sharing common signaling components, we find that the response profiles of the NFkB and MAPK signaling modules vary with respect to kinetics, response magnitude and pathway selectivity. Analysis of the response to combined stimuli (mimicking what would occur with an intact pathogen) leads to non-additive levels of activation of downstream signaling pathways. In collaboration with the PSIIM Computational Biology group, we will use these observations to develop quantitative simulations of single and multi-receptor activation in an effort to understand the non-additive outputs observed in our experiments. These models will be used as a basis for understanding the interplay between PRR-activated pathways during an infection and the subsequent host response. We have also initiated a specific study of the macrophage response to Burkholderia cenocepacia (Bcc), an opportunistic bacteria particularly problematic in cystic fibrosis and chronic granulomatous disease patients, and closely related to the category A select agents Burkholderia mallei and pseudomallei. Macrophages are likely to play a key role in Bcc-induced pulmonary infections, but very little is known about the mechanism of Bcc infection and replication in these cells. We have studied the infection of human monocytic cells with virulent (J2315) and less virulent (K56-2) strains of Bcc to characterize growth kinetics, cytotoxicity, intracellular trafficking and induction of cellular responses such as autophagy and apoptosis. To determine the contribution of TLR signaling responses to infection, we have compared the ability of live and formalin killed bacteria to initiate early signaling responses and later secretion of a range of cytokines. In addition to providing important insight to the macrophage response to this pathogen in clinically relevant circumstances, the model described earlier for combined TLR activation will be used in an effort to determine the relative contribution of different TLR pathways to the host response to Bcc, and possibly gain insight to the mechanism of virulence adopted by this pathogen. In a continuation of a collaborative project with colleagues from the Alliance for Cellular Signaling, we published a study on the subversion of Ca2+ signaling pathways in macrophages by Clostridium difficile, a major cause of acute colitis. We determined that the bacterial toxin, ToxB, specifically attenuated Ca2+ signaling through one class of G-protein coupled receptor while enhancing signaling through another, which may have a key role in altering the inflammatory state of macrophages resident in the gut mucosa.